Htb 43

The study was conducted using three HNSCC cell lines: H103 (ECACC, no. 06092001), FaDu (ATCC^®, HTB-43™), and HPV positive—KB (ATCC^®, CCL-17™). FaDu and KB cell lines were cultured in DMEM (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (Biowest, France) and 1% of penicillin and streptomycin mixture (Merck, Darmstadt, Germany); H103 cells were cultured in 1:1 DMEM and Ham’s F12 (Biowest, France) nutrient medium with 10% fetal bovine serum, 1% of L-glutamine (Biowest, France) and 1% of penicillin and streptomycin mixture. All cells were cultured at 37 °C with 100% humidity at the 5% CO2 atmosphere.

The human HNSC cell lines FaDu (human hypopharynx cancer cells, ATCC HTB-43) and SCC-9 (human oral cavity cancer cells, ATCC CRL-1629) were purchased from the ATCC. All media were added with 10% fetal bovine serum (FBS) (Minhai Bio-engineering Co., LTD) and 1% penicillin–streptomycin solution (Beyotime). All cell lines were cultured within 2 months of resuscitation under standard conditions at 37 °C, 5% CO₂. FaDu cell line was cultured in MEM, and SCC-9 cell line was cultured in DMEM.

Human colorectal HCT-116 and hypopharyngeal FaDu cancer cell lines were purchased from ATCC (CCL-247 and HTB-43). Cell lines were stored in liquid nitrogen in DMEM supplemented with 10% heat-inactivated FBS (Sigma, Saint-Louis, MO, USA) and 5% DMSO (Sigma, Saint-Louis, MO, USA). All cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 10 mM D-glucose, 2 mM L-glutamine and 25 mM of PIPES and HEPES and adjusted to pH 7.4 with NaOH 5 M (Merck, Darmstadt, Germany). All cell lines tested negative for mycoplasma contamination using the MycoAlert^TM Mycoplasma Detection kit (Lonza, Basel, Switzerland).

The human squamous cell carcinoma cell line FaDu (obtained from ATCC; ATCC^® HTB43™) and the murine colon carcinoma CT26.WT (hereinafter referred to as CT26) tumor cell line (obtained from ATCC, Manassas, VA, USA; ATCC^® CRL-2638™) were cultured in the ATCC-suggested cell culture mediums. The FaDu cells were cultured in Advanced Dulbecco’s Modified Eagle’s Medium (A-DMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and the CT26 cells were cultured in Advanced RPMI-1640 (A-RPMI, Gibco), both supplemented with 2 mM L-glutamine (Gibco), 5% (v/v) fetal bovine serum (FBS, Gibco), GlutaMAX (Gibco) and 1% (v/v) Penicillin–Streptomycin (stock solution, 10,000 U/mL, Gibco). Additionally, HEK-Blue™ IL-12 cells (IL-12 reporter cells) (InvivoGen) were used to determine the biological activity and potency of phIL12 and pmIL12. The HEK-Blue™ IL-12 cells were cultured in A-DMEM (Gibco) supplemented with 2 mM L-glutamine (Gibco), 5% (v/v) FBS (Gibco) and 1% (v/v) Penicillin–Streptomycin (stock solution, 10,000 U/mL, Gibco). After the second passage, the 1× HEK-Blue™ Selection (InvivoGen) was added to the growth medium. The cells were handled according to the supplier’s instructions and cultured in a 5% CO₂ humidified incubator at 37 °C. For the experiments, the cells were maintained in monolayers until they reached 70–80% confluence.

Human hypopharyngeal cancer FaDu cells (ATCC^® HTB-43™) and human skin fibroblast Detroit 551 cells (ATCC^® CCL-110™) were cultured in Eagle’s minimum essential medium (Thermo Fisher Scientific, GIBCO, Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone, UT, USA), 1% penicillin–streptomycin (Thermo Fisher Scientific, GIBCO, Waltham, MA, USA), 1% non-essential amino acid, and 1% sodium pyruvate at 37 °C in an atmosphere containing 5% CO₂. Cells were harvested after 48 h in metformin (Sigma-Aldrich, St. Louis, MO, USA) treatment. For protein expression analysis of signaling pathways, the cells were pretreated with the inhibitors of PD98059 (Calbiochem, San Diego, CA, USA) or SB203580 (Calbiochem, San Diego, CA, USA) for 1 h.

Human HNC-SCC cell lines were purchased from the American Tissue Culture Collection (ATCC). FaDu (ATCC^® HTB-43™) was derived from the pharynx, and both CAL 27 (ATCC^® CRL-2095™) and UPCI: SCC154 (which is HPV positive) (ATCC^® CRL-3241™) were from the tongue. Cells were incubated at 37 °C and 5% CO₂. Eagle’s Minimum Essential Medium (BD, USA) was used to culture FaDu and SCC154, and Dulbecco’s Modified Eagle’s Medium (Lonza, Slough, UK/Corning, Manassas, VA, USA) was used for CAL27. The media were supplemented with foetal bovine serum (10%) (Biosera, Cholet, France), L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Gibco, Waltham, MA, USA). The culture medium was replaced three times a week, and cells were checked regularly.

CAL27 (CRL-2095) was obtained from ATCC and maintained in Dulbecco’s minimal essential medium (DMEM). HTB-43 was obtained from ATCC and maintained in ATCC-formulated Eagle’s Minimum Essential Medium (EMEM). HCSS-4 (CRL-1582) was obtained from ATCC and maintained in ATCC-formulated RPMI-1640 Medium. THP-1 (TIB-202™) was obtained from ATCC and maintained in ATCC-formulated RPMI. RAW 264.7 macrophages (ATCC TIB-71) were obtained from ATCC and cultured in DMEM. All cells were supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin and incubated at 37 °C in 5% CO₂.